Expression cassette and vector for transient or stable expression of exogenous molecules

ABSTRACT

The disclosure provides an expression cassette and a vector comprising the cassette for expression of a polynucleotide. The expression cassette includes a promoter/enhancer, an intervening region, and a polyadenylation signal domain. Expression systems and methods of using the expression cassette and vector are also provided.

TECHNICAL FIELD

This invention relates to expression vectors and expression cassettes,and more particularly to methods, compositions, and systems forexpression of an exogenous molecule in an organism.

BACKGROUND

The introduction of nucleic acid molecules, polypeptide, peptides, andsmall molecules into target cells and tissues is being used both as atherapeutic delivery system as well as in the production of therapeuticmolecules in vitro. The applicability of this approach has increasedwith the further understanding of host cells and the molecular biologyof cell division, differentiation, and expression mechanisms.

SUMMARY

It has been discovered that transcription driven by a CMV promoter andterminated by a polyA domain from a variant human growth hormone (hGHv)gene is more efficient than other expression vectors lacking one or theother or both such elements. Therefore, the invention provides anexpression cassette and an expression vector useful in the expression ofpolynucleotides of interest. The expression cassette of the inventionincludes a combination of regulatory elements that provide efficienttranscription, efficient transcription termination, and increased mRNAstability of transcribed products. In one embodiment, the expressioncassette includes a human cytomegalovirus promoter/enhancer, a cloningsite or polynucleotide of interest, and a hGHv polyadenylation signaldomain. Optionally a variable length intervening sequence may bepresent.

The invention provides an expression cassette that includes a human CMVimmediate early 1 (hCMV IE1) promoter/enhancer region, a polynucleotideof interest, and a variant human growth hormone (hGHv) polyA signaldomain or variant thereof. The polyA signal variant is at least 100nucleotides in length and contains the sequence AATAAA, and is at least92% identical to a hGH polyA signal domain.

The invention further provides an expression vector that includes anexpression cassette of the invention as well as host cells containing aexpression cassette or expression vector of the invention.

The invention further provides an expression cassette that includes ahuman CMV immediate early 1 (hCMV IE1) promoter/enhancer region, avariable length intervening sequence (VLIVS) comprising a splice donorand splice acceptor site, a polynucleotide of interest, and a varianthuman growth hormone (hGHv) polyA signal domain or variant thereof. ThepolyA signal domain or variant thereof is at least 100 nucleotides inlength and contains the sequence AATAAA and is at least 92% identical toa hGHv polyA signal domain.

The invention also provides an expression vector that includes a humanCMV immediate early 1 (hCMV IE1) promoter/enhancer region, a variablelength intervening sequence (VLIVS) comprising a splice donor site and asplice acceptor site, a cloning site, a hGH poly adenylation region, anda selectable marker. In one aspect of the invention, the hCMV IE1promoter/enhancer region is upstream (5′) to the cloning site and thehGH poly adenylation region is downstream (3′) to the cloning site.

The invention also includes a method of delivering an agent of interestin vivo. The method includes delivering a composition comprising anexpression cassette to a subject, the expression cassette includes ahCMV IE1 promoter/enhancer region; a variable length interveningsequence comprising a splice donor and splice acceptor site; apolynucleotide encoding the agent of interest; and a human growthhormone (hGH) polyA signal domain or variant thereof.

The invention further includes an expression system. The expressionsystem includes a host cell transfected or transformed with anexpression cassette of the invention, wherein the host cell is culturedunder conditions to express the polynucleotide of interest; andrecovering the agent of interest.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. All references citedherein are incorporated by reference.

DESCRIPTION OF DRAWINGS

FIG. 1 is a plasmid map of the pV10 vector. The cytomegalovirusimmediate early 1 (CMV IE1) promoter/intron (IVS) was generated by PCRand cloned into the HindIII and BamH1 sites. The ampicillin resistancegene, beta lactamase (bla), is also indicated.

FIG. 2 is a plasmid map of pV40. Indicated are the cytomegalovirusimmediate early 1 (CMV IE1) promoter, an intron (IVS), a hGHvpolyadenylation signal domain (polyA) of about 600 base pairs in length,and the ampicillin resistance gene, beta lactamase (bla).

FIG. 3 is a schematic representation of the plasmid pV70. Indicated arethe CMV IE1 promoter, the IVS including the deletion junction and thesplice donor (SD) and splice acceptor (SA) sites, a hGHv polyA signaldomain, and the bla gene. The deletion junction represents theblunt-ended ligation result of BspE1′/′HpaI.

FIG. 4 is a schematic representation of the generation of the pXLC.1vector. The pXLC.1 vector was constructed using the expression vectorpV70 and a PCR product containing the light chain coding sequence. PV70was linearized with BamHI, the PCR product was digested with BamHI andthe two were ligated together to generate the pXLC.1 vector.

FIG. 5 is a schematic representation of the generation of the pXLC.2vector.

FIG. 6 is a schematic representation of a pV60 vector and the generationof the pXHC vector. The pXHC vector was constructed using the expressionvector pV60 and a PCR product containing most of the heavy chain codingsequence (52 amino acids from the N-terminal were not included). PV60was linearized with BamHI, the PCR product was digested with BamHI andthe two were ligated together.

FIG. 7 is a schematic representation of a pXHC.1 vector.

FIG. 8 is a schematic representation of a pXHC.3 vector.

FIG. 9 is a schematic representation of a pXHC.5 vector, resulting fromthe addition of a dhfr cassette to pXHC.3. The control elements of theexpression cassette were derived from pSI (Promega, Genbank accession#U47121) and include the SV40 promoter/enhancer, an artificial intronand the SV40 late polyadenylation sequence.

FIG. 10 is a schematic representation of the vector pV80. Indicated arethe cytomegalovirus immediate early 1 (CMV IE1) promoter/intron (IVS)fragment including the splice donor (SD) and the splice acceptor (SA)sites, a hGHv polyadenylation signal domain (polyA), the ampicillinresistance gene, beta lactamase (bla), the SV40 promoter/enhancer theartificial intron and the SV40 late polyadenylation sequence.

FIGS. 11A and 11B are schematic representations of the vector pV90 andthe corresponding annotated sequences. FIG. 11A is a vector map.Indicated are cytomegalovirus immediate early 1 (CMV IE1)promoter/intron (IVS) fragment including the splice donor (SD) and thesplice acceptor (SA) sites, a hGHv polyadenylation signal domain(polyA), the ampicillin resistance gene, beta lactamase (bla), the SV40promoter/enhancer, the artificial intron and the SV40 latepolyadenylation sequence. The vector lacks the NotI site in the dhfrexpression cassette. FIG. 11B is the annotated sequence of the pV90vector. In FIG. 11B, the sequence from nucleotides 1275 to 1866 of SEQID NO:19 represents a hGHv polyA of about 600 nucleotides in length.

FIG. 12 is a vector map of pSI-DHFR.2. The SV40 promoter drives the dhfrgene.

FIG. 13 is a graph depicting the relative specific productivities of thetransfected pools. Three pools were analyzed for pCMV-hGHvPA-SEAP andpSEAP2, and one for pUC18.

FIG. 14 is a pair of graphs showing the relative specific productivitiesof the isolates. The top twenty isolates from each construct are shownin rank order.

FIG. 15 shows a sequence in GenBank Accession No. K00470 (SEQ ID NO:18).

DETAILED DESCRIPTION

The invention relates to an expression cassette and vectors containingthe expression cassette. The expression cassette includes atranscriptional regulatory region capable of driving transcription ineukaryotic host and a transcriptional termination region. The expressioncassettes and vectors of the invention provide for a strongtranscription start and stop as well as increased mRNA stability oftranscribed products.

The invention provides promoter and optionally enhancer elements fromany strain of cytomegalovirus, such as described herein or in referencessuch as U.S. Pat. No. 5,658,759, the disclosure of which is incorporatedherein by reference. For example, suitable CMV immediate early promoterregions useful in the expression cassettes of the invention can beobtained from the CMV-promoted β-galactosidase expression vector, CMVβ(MacGregor et al., Nucl. Acids Res. 17:2365 (1989)).

As discussed further herein, the hGHv polyadenylation signal domainprovides a strong transcriptional stop signal as well as increases thestability of the mRNA transcript. The regulatory/expression element maybe separated from the hGHv polyadenylation signal domain by, forexample, a polynucleotide of interest or a cloning site (e.g., amultiple cloning site).

In one aspect of the invention, there is provided a polynucleotidecomprising an expression cassette that includes a cytomegalovirus (CMV)transcriptional regulatory region, a variable length interveningsequence (e.g., from intron A of CMV), a polynucleotide of interest, anda polyadenylation signal domain. The invention further relates toprocesses and expression vectors for producing and recoveringheterologous polypeptides from host cells.

In another aspect, an expression cassette of the invention includes,operably linked, (i) a CMV major immediate early 1 (IE1)promoter/enhancer region and a variable length intervening sequence(e.g., derivative of intron A), (ii) a polynucleotide of interest, and(iii) a hGHv polyadenylation signal domain. The term “operably linked”refers to a juxtaposition wherein the components are in a relationshippermitting them to function in their intended manner (e.g., functionallylinked). Thus, for example, a promoter/enhancer operably linked to apolynucleotide of interest is ligated to the latter in such a way thatexpression of the polynucleotide of interest is achieved underconditions which are compatible with the activation of expression fromthe promoter/enhancer.

In a specific embodiment of the invention, the expression cassetteincludes a sequence as set forth in SEQ ID NO:1 from about nucleotide 1to about nucleotide 1867 (e.g., from about 1 to 1865, 1866, 1867, 1868,or 1869). The expression cassette set forth from nucleotide 1 to 1867 ofSEQ ID NO:1 includes a number of distinct domains such as a CMV IE1promoter/enhancer region having a sequence as set forth from about x₁ toabout x₂ of SEQ ID NO:1, wherein x₁ is a nucleotide from 1-20 and x₂ isa nucleotide from about 715-720 (e.g., from about 1 to 719 of SEQ IDNO:1). Another domain of the expression cassette includes a variablelength intervening sequence (VLIVS) containing a splice donor and asplice acceptor site. The VLIVS can be at least 50 by in length (e.g.,at least 100, 150, 200, or 250 by in length) and can include splicedonors and acceptors from any source known in the art. See, e.g., Varaniet al., Annu Rev Biophys Biomol Struct 27:407-45 (1998) and Koning, EurJ Biochem 219:25-42 (1994). A suitable intervening domain can includeall of intron A of a CMV genome of any strain or may include a smallerfragment comprising a 5′ sequence containing a splice donor site ligatedto a 3′ sequence containing a splice acceptor site. For example, theVLIVS includes nucleotides from about x₃ to about x₄ of SEQ ID NO:1,wherein x₃ is a nucleotide from 715-720 and x₄ is a nucleotide from1236-1254 (e.g., 719 to 1236 of SEQ ID NO:1). The intervening sequencefollowing the CMV IE1 promoter/enhancer can vary in size as much as 317nucleotides from that present in SEQ ID NO:1. For example, 317nucleotides were deleted from the IVS sequence as depicted in pV40 andpV70 (see, e.g., FIGS. 2 and 3, respectively) to produce the VLIVS ofSEQ ID NO:1. Thus, in another aspect of the invention the expressioncassette includes a sequence from about nucleotide 1 to about 1254 ofSEQ ID NO:1 (e.g., a CMV IE1 promoter/enhancer and an interveningsequence). A multiple cloning site may be present after (i.e.,downstream of) the IVS region (e.g., nucleotides 1255-1272 of SEQ IDNO:1 includes BamH1 sites and a Not1 site). Different or additionalrestriction sites may be engineered in the expression cassette usingtechniques known to those of skill in the art. The expression cassettefurther includes a polyA domain.

The polyA signal domain is derived from a hGHv gene, which can vary inits 3′UTR sequence, e.g., from allele to allele. One allele of the hGHvgene is described in GenBank Accession No. K00470 (SEQ ID NO:18), whileanother sequence is described in FIG. 11B as SEQ ID NO:19, whichcorresponds to nucleotides 2032 to 2625 of SEQ ID NO:18 (See FIG. 15).Non-naturally occurring variants of the polyA signal domain may be madeby mutagenesis techniques, including those applied to polynucleotides,cells or organisms. A polyA variant from a hGHv gene includes polyAsignal domain that varies from a wild-type hGHv polyA signal domain yetretains the ability to signal transcriptional termination and/orstabilize mRNA. For example, the polyadenylation signal domain mayinclude an hGHv polyadenylation signal domain sequence as set forth inSEQ ID NOs:18 or 19. One skilled in the art of molecular biology willalso understand that the sequences need not be as long as about 600nucleotides. Rather, any polyA sequence domain that includes acontiguous nucleotide sequence of at least 100 nt (e.g., at least 200,300, 400, 500, or 600 nt), including the canonical AATAAA site, of ahGHv gene is included. In addition, the invention encompasses sequencesthat vary from the foregoing sequences by up to 8% (e.g., have 92%identity to SEQ ID NO:18 or 19 or a distinct domain thereof). Forexample, a polynucleotide of 100 nt in length having 95% identity tonucleotides 1-1867 of SEQ ID NO:1 and including the sequence AATAAAwould retain the ability to terminate transcription.

In another aspect of the invention a vector comprising an expressioncassette is provided. As used herein, a “vector” is a nucleic acidmolecule (either DNA or RNA) capable of autonomous replication uponintroduction into a recipient cell (e.g., a bacterium such as E. coli).Plasmids, viruses and bacteriophages are examples of vectors. Theprocess of “expression” from an expression vector is well known, andincludes the use of cellular enzymes and processes to produce anexpression product from a polynucleotide of interest. Expression vectorsare vectors that are capable of mediating the expression of a clonedpolynucleotide in a host cell; which may or may not be the same type ofcell used for replication or propagation of the vector. Many mammalianexpression vectors can be propagated in common bacteria (recipient cell)but express the polynucleotide of interest in mammalian cells (hostcell) and not in bacterium.

The invention concerns the design and use of vectors that are capable ofpermitting efficient transcription and translation of polynucleotides ineukaryotic (e.g., mammalian, and most particularly, human, murine,simian, bovine, porcine, rodent, or ovine cells) cells. The vectors ofthe invention include an expression cassette as set forth aboveincluding a polyadenylation signal domain that provides for efficienttranscriptional termination and mRNA stability.

The vectors of the invention include: a cloning site for receiving apolynucleotide of interest; transcription regulatory elements (e.g., CMVIE1 promoter/enhancer regions) sufficient to permit transcription of apolynucleotide inserted into the cloning site in a host cell;translation elements sufficient to permit translation of an RNAtranscript of said polynucleotide in a host cell and (if desired)replication elements sufficient to permit the replication of said vectorin a host cell or another recipient cell used for propagation of thevector. The vectors of the invention are capable of mediating suchexpression transiently or stably in host cells.

In a specific embodiment a vector of the invention includes (1) asequence as set forth in SEQ ID NO:1; (2) a sequence that iscomplementary to the sequence as set forth in SEQ ID NO:1; (3) asequence that is at least 80% (preferably at least 90%; 95%; 98% or 99%)identical to SEQ ID NO:1 or its complement; or (4) a vector comprisingSEQ ID NO:1 from about nucleotide 1 to about nucleotide 1867 andcomprising a polynucleotide of interest and/or a selectable marker.

The vector comprising SEQ ID NO:1 has a number of distinct domains andcoding regions. For example, a CMV IE1 promoter/enhancer region having asequence as set forth from about x₁ to about x₂ of SEQ ID NO:1, whereinx₁ is a nucleotide from 1-20 and x₂ is a nucleotide from about 715-720(e.g., from about 1 to 719 of SEQ ID NO:1) is present in the vector.Another domain of an expression vector of the invention includes avariable length intervening sequence (VLIVS) containing a splice donorand splice acceptor site. For example, the IVS includes nucleotides fromabout x₃ to about x₄ of SEQ ID NO:1, wherein x₃ includes a nucleotidefrom 715-720 and x₄ includes a nucleotide from 1236-1254 (e.g., aboutnucleotides 719 to 1236 of SEQ ID NO:1). A multiple cloning site of theexpression vector includes nucleotides 1255-1272 of SEQ ID NO:1 (e.g.,BamH1 sites and a Not1 site). Different or additional restriction sitesmay be engineered in the expression vector using techniques known tothose of skill in the art. The expression vector further includes apolyA signal domain. The polyA signal domain is a hGHv polyA signaldomain or other variant of the hGH polyA signal domain. For example, apolyA signal domain includes an hGHv polyA signal domain sequence as setforth in SEQ ID NO:19. Also present in a vector of the invention is oneor more selectable markers. For example, SEQ ID NO:1 includes adihydrofolate reductase (dhfr) gene (e.g., from about nucleotide 2568 toabout nucleotide 3132 of SEQ ID NO:1). A vector of the invention mayinclude additional promoter/enhancer elements and regulatory regions(e.g., polyadenylation domains) in addition to those provided above.Such additional regulatory elements and polyadenylation domains mayflank (e.g., be immediately adjacent to, 5′and 3′ of) a selectablemarker or polynucleotide of interest. For example, the vector comprisingSEQ ID NO:1 contains a dihydrofolate reductase (dhfr) gene from aboutnucleotide 2568 to about nucleotide 3132 of SEQ ID NO:1. The dhfr geneis flanked by an SV40 promoter/enhancer element and an SV40polyadenylation region (e.g., about nucleotide 1868 to about nucleotide2210 and about nucleotide 3144 to about nucleotide 3440 of SEQ ID NO:1,respectively).

Specific examples of selectable markers are those that encode proteinsthat confer resistance to cytostatic or cytocidal drugs, such as theDHFR protein; which confers resistance to methotrexate ogler et al.,Proc. Natl. Acad. Sci. USA 77:3567 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); the GPF protein, which confersresistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.USA 78:2072 (1981)), the neomycin resistance marker, which confersresistance to the aminoglycoside G-418 (Colberre-Garapin et al., J. Mol.Biol. 150:1 (1981)); the Hygro protein, which confers resistance tohygromycin (Santerre et al., Gene 30:147 (1984)); and the Zeocin™resistance marker (available commercially from Invitrogen). In addition,the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Other selectablemarkers encode puromycin N-acetyl transferase or adenosine deaminase.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid molecules, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence over a comparison window, as measured using a comparisonalgorithm or by manual alignment and visual inspection. This definitionalso refers to the complement of a sequence (e.g., the complement of asequence as set forth in SEQ ID NO:1 or a fragment thereof comprising anexpression cassette). For example, the expression cassette and fragmentsthereof include those with a nucleotide sequence identity that is atleast about 80%, about 90%, and about 95%, about 97%, about 98% or about99% identical to a portion of SEQ ID NO:1 (e.g., nucleotides 1-719,1-1254, and the like, of SEQ ID NO:1). Thus, if a sequence has therequisite sequence identity to the full sequence of SEQ ID NO:1 or adomain thereof then it can also function as an expression cassette ordomain of the invention, respectively.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentity for the test sequence(s) relative to the reference sequence,based on the designated or default program parameters. A “comparisonwindow”, as used herein, includes reference to a segment of any one ofthe number of contiguous positions selected from the group consisting offrom 25 to 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, PILEUP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection.

One example of an algorithm that is suitable for determining percentsequence identity (i.e., substantial similarity or identity) is theBLAST algorithm, which is described in Altschul, J. Mol. Biol.215:403-410, 1990. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information (onthe World Wide Web at ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. “T” is referred to as theneighborhood word score threshold. These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always>0)and N (penalty score for mismatching residues, always<0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. In oneembodiment, to determine if a nucleic acid sequence is within the scopeof the invention, the BLASTN program (for nucleotide sequences) is usedincorporating as defaults a wordlength (W) of 11, an expectation (E) of10, M=5, N=4, and a comparison of both strands. For amino acidsequences, the BLASTP program uses as default parameters a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix(see, e.g., Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989).

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin, Proc. Nat'l. Acad.Sci. USA 90:5873-5787, 1993). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Also included in the invention are polynucleotides that specificallyhybridize to a polynucleotide sequence as set forth in SEQ ID NO:1 fromabout nucleotide 1 to 1867 or a fragment thereof. The phrase“selectively (or specifically) hybridizes to” refers to the binding,duplexing, or hybridizing of a molecule to a particular referencepolynucleotide under stringent hybridization conditions. The phrase“stringent hybridization conditions” refers to conditions under which aprobe will primarily hybridize to its target subsequence, typically in acomplex mixture of nucleic acid, but to no other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances, e.g., depending on the length of the probe. Longersequences hybridize specifically at higher temperatures. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Techniques in Biochemistry and Molecular Biology—Hybridization withNucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength, pH, andnucleic concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 M sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g., 10 to about 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than about 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal(e.g., identification of a nucleic acid of the invention) is about 2times background hybridization. “Stringent” hybridization conditionsthat are used to identify substantially identical nucleic acids withinthe scope of the invention include hybridization in a buffer comprising50% formamide, 5×SSC, and 1% SDS at a temperature between 42° C. and 65°C., with a wash of 0.2×SSC and 0.1% SDS at 65° C., for long probes.However, as is apparent to one of ordinary skill in the art,hybridization conditions can be modified depending on sequencecomposition. Exemplary “moderately stringent hybridization conditions”include a hybridization in a buffer of 40% formamide, 1 M NaCl, and 1%SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization isat least twice background. Those of ordinary skill will readilyrecognize that alternative hybridization and wash conditions can beutilized to provide conditions of similar stringency. Thus, anexpression cassette of the invention can include a hGHv polyA signaldomain that hybridizes under high stringency conditions to a ssDNAcontaining the nucleotide sequence of SEQ ID NO:18 or 19.

The expression cassette may be used in the form of a naked nucleic acidconstruct. Alternatively, the expression cassette may be introduced aspart of a nucleic acid vector (e.g., an expression vector such as thosedescribed above). Such vectors include plasmids and viral vectors. Avector may include sequences flanking the expression cassette thatinclude sequences homologous to eukaryotic genomic sequences, such asmammalian genomic sequences, or viral genomic sequences. This will allowthe introduction of the expression cassette into the genome ofeukaryotic cells or viruses by homologous recombination. For example, aplasmid vector comprising the expression cassette flanked by viralsequences can be used to prepare a viral vector suitable for deliveringthe expression cassette to a vertebrate, including fish, avian ormammalian cells. The techniques employed are well known to a skilledperson.

The term “polynucleotide of interest” is intended to cover nucleic acidmolecules that are capable of being at least transcribed. The moleculemay be in the sense or antisense orientation with respect to thepromoter. Antisense constructs can be used to inhibit the expression ofa gene in a cell according to well-known techniques. The polynucleotideof interest may include a heterologous polynucleotide. The termheterologous polynucleotide encompasses any gene. A heterologouspolynucleotide typically originates from a foreign species compared tothe regulatory element with which it is operably linked in theexpression cassette or vector or if originated from the same source, isthe modified gene from its original form. Therefore, a heterologouspolynucleotide operably linked to a promoter is from a source differentfrom that from which the promoter was derived, or, if originated fromthe same source, is the modified promoter from its original form.Modification of the heterologous polynucleotide may occur, e.g., bytreating the DNA with a restriction enzyme to generate a DNA fragmentthat is capable of being operably linked to the promoter. Site-directedmutagenesis is also useful for modifying a heterologous polynucleotide.Heterologous polynucleotides may also include marker genes (e.g.,encoding β-galactosidase or green fluorescent protein) or genes whoseproducts regulate the expression of other genes. Thus polynucleotidesthat serve as templates for mRNA, tRNA and rRNA are included within thisdefinition. The heterologous gene may be any allelic variant of awild-type gene, or it may be a mutant gene. mRNA will optionally includesome or all of 5′ and/or 3′ transcribed but untranslated flankingregions naturally, or otherwise, associated with the translated codingsequence.

The polynucleotide of interest may optionally further include theassociated transcriptional control elements normally associated with thetranscribed molecules, for example transcriptional stop signals,polyadenylation domains and downstream enhancer elements. Thepolynucleotide of interest can encode or serve as template for atherapeutic product, which can for example be a peptide, polypeptide,protein, or ribonucleic acid. The polynucleotide of interest istypically a DNA sequence (such as cDNA or genomic DNA) coding for apolypeptide product such as enzymes (e.g. β-galactosidase); bloodderivatives; hormones; cytokines; interleukins; interferons; TNF; growthfactors (e.g. IGF-1); soluble receptor molecules (e.g., soluble TNFreceptor molecules); neurotransmitters or their precursors; trophicfactors such as BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3 and NT5;apolipoproteins such as ApoAI and ApoAIV; dystrophin or aminidystrophin; tumor-suppressing proteins such as p53, Rb, Rap1A, DCCand k-rev; factors involved in coagulation such as factors VII, VIII andIX; or alternatively all or part of a natural or artificialimmunoglobulin (e.g. Fab and ScFv, or the light or heavy chain of acloned IgG).

A polynucleotide of interest may also include a template for generationof an antisense molecule, whose transcription in a target cell enablesgene expression or the transcription of cellular mRNAs to be controlled.Such molecules can, for example, be transcribed in a target cell intoRNAs complementary to cellular mRNAs and can thus block theirtranslation into protein, according to techniques known in the art. Inparticular, antisense molecules can be used to block translation ofinflammatory or catabolic cytokines in the treatment of arthritis andtissue loss caused by these cytokines.

The polynucleotide sequence of interest typically will encode apolypeptide of diagnostic or therapeutic use. The polypeptide may beproduced in bioreactors in vitro using various host cells (e.g., COScells or CHO cells or derivatives thereof) containing the expressioncassette of the invention. Alternatively, the expression cassette and/orvector of the invention may be used for gene delivery, protein delivery,and/or gene therapy.

The invention may also be used for the expression of toxic factors andpolypeptides. The latter can be, in particular, cell poisons (such asdiphtheria toxin, pseudomonas toxin and ricin A), a product inducingsensitivity to an external agent (e.g. thymidine kinase and cytosinedeaminase) or alternatively factors capable of inducing cell death (e.g.Grb3-3 and anti-ras ScFv).

By a therapeutic use is meant a use that may provide relief from adisease or disorder, cure a disease or disorder, and/or ameliorate theseverity of a disease or disorder. A diagnostic use includes usingmolecules capable of determining or providing information regarding acause or relationship of a molecule to a disease process or determiningthe presence or absence of a disease or disorder. A diagnostic agentdoes not directly contribute to the amelioration of the disease ordisorder.

A polynucleotide of interest may also encode an antigenic polypeptidefor use as a vaccine. Antigenic polypeptides or nucleic acid moleculesare derived from pathogenic organisms such as, for example, a bacteriumor a virus. For example, antigenic polypeptides include antigenicdeterminants present in the genomes or gene products of a pathogenicorganism, for example, viral haemorrhagic septicemia, bacterial kidneydisease, vibriosis, and furunculosis. Antigenic polypeptides may beselected from regions of the hepatitis C virus genome and gene products,for example.

As used herein, “isolated,” when referring to a molecule or composition,such as, e.g., a vector or expression cassette of the invention, orpolynucleotide of interest, means that the molecule or composition isseparated from at least one other compound, such as a protein, DNA, RNA,or other contaminants with which it is associated in vivo or in itsnaturally occurring state. Thus, a polynucleotide of interest isconsidered isolated when it has been isolated from any other componentwith which it is naturally associated. An isolated composition can,however, also be substantially pure. An isolated composition can be in ahomogeneous state. It can be in a dry/lyophilized or an aqueoussolution. Purity and homogeneity can be determined, e.g., usinganalytical chemistry techniques such as, e.g., polyacrylamide gelelectrophoresis (PAGE), agarose gel electrophoresis or high-pressureliquid chromatography (HPLC).

As used herein, the terms “nucleic acid molecule” and “polynucleotide”are used interchangeably, and include oligonucleotides (i.e., shortpolynucleotides). They also refer to synthetic and/or non-naturallyoccurring nucleic acid molecules (e.g., comprising nucleotide analoguesor modified backbone residues or linkages). The terms also refer todeoxyribonucleotide or ribonucleotide oligonucleotides in eithersingle-or double-stranded form. The terms encompass nucleic acidscontaining analogues of natural nucleotides. The terms also encompassnucleic acid-like structures with synthetic backbones. DNA backboneanalogues provided by the invention include phosphodiester,phosphorothioate, phosphorodithioate, methyl-phosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholino carbamate, andpeptide nucleic acids (FNAs); see Oligonucleotides and Analogues, aPractical Approach, edited by F. Eckstein, IRL Press at OxfordUniversity Press (1991); Antisense Strategies, Annals of the New YorkAcademy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992);Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research andApplications (1993, CRC Press). PNAs contain non-ionic backbones, suchas N-(2-aminoethyl)glycine units. Phosphorothioate linkages aredescribed in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl.Pharmacol. 144:189-197. Other synthetic backbones encompassed by theterm include methyl-phosphonate linkages or alternatingmethylphosphonate and phosphodiester linkages (Strauss-Soukup (1997)Biochemistry 36:8692-8698), and benzyl-phosphonate linkages (Samstag(1996) Antisense Nucleic Acid Drug Dev 6:153-156).

As used herein, “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (e.g., “recombinant polynucleotide”), tomethods of using recombinant polynucleotides to produce products incells or other biological systems, or to a polypeptide (“recombinantprotein”) encoded by a recombinant polynucleotide. Recombinantpolynucleotides encompass nucleic acid molecules from different sourcesligated into an expression cassette or vector for expression of, e.g., afusion protein; or those produced by inducible or constitutiveexpression of a polypeptide (e.g., an expression cassette or vector ofthe invention operably linked to a heterologous polynucleotide, such asa polypeptide coding sequence).

In a typical expression system, production of a polypeptide from aheterologous polynucleotide is either not regulated or is regulated bymodulating transcription from a transcriptional promoter operably linkedupstream of a polynucleotide that encodes the heterologous polypeptide.However, regulation must also occur properly downstream in order provideproper transcriptional termination and mRNA stability. In one aspect ofthe invention, a human growth hormone variant (hGHv) polyadenylation(polyA) signal domain is provided downstream (3′) of a polynucleotide ofinterest present in an expression cassette or vector of the invention.The hGHv polyA signal domain includes a sequence derived from the humangrowth hormone genetic sequence. The hGHv polyadenylation signal domainsequence provides for a strong transcriptional termination and providesincreased mRNA stability in eukaryotic cells. This hGHv polyadenylationsignal domain provides a distinctive advantage over prior expressioncassettes and/or vectors including those that may utilize a CMVpromoter/enhancer.

Translation elements may also be present and are intended to encompassthe specialized sequences (such as ribosome binding sites and initiationcodons) that are necessary to permit translation of an RNA transcriptinto protein. Translation elements may also include consensus sequences,leader sequences, splice signals, and the like, that serve to facilitateor enhance the extent of translation, or increase the stability of theexpressed product. For example, the hGHv polyadenylation signal domainprovides increased mRNA stability. The vectors of the invention maypossess ancillary transcription regions, such as introns,polyadenylation signals, Shine/Dalgarno translation signals and Kozakconsensus sequences (Shine et al., Proc. Natl. Acad. Sci. (U.S.A.)71:1342-1346 (1974); Kozak, Cell 44:283-292 (1986)).

The term “replication elements” is intended to encompass the specializedsequences (such as origins of replication) that are necessary to permitreplication of the vector in a recipient cell. In general, such vectorswill contain at least one origin of replication sufficient to permit theautonomous stable replication of the vector in a recipient cell.

To facilitate selection and maintenance of a vector of the invention,one or more selectable markers (such as polynucleotides that conferresistance to antibiotics, or a cellular capacity to grow on minimalmedium or in the presence of toxic metabolites) may be included in thevector.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs (e.g., the expression cassetteor vector of the invention). The expression cassette of the inventionmay be used to recombinantly modify a host cell by transfecting a hostcell or transforming a host cell to express a desired polynucleotide ofinterest. As used herein, the term “recombinantly modified” meansintroducing an expression cassette or vector of the invention into aliving cell or expression system. Usually, the expression cassettecomprising a polynucleotide of interest is present in a vector (e.g., aplasmid). An expression system includes a living host cell into which apolynucleotide of interest, whose product is to be expressed, has beenintroduced, as described herein.

Host cells are cells in which an expression cassette (including a vectorcomprising an expression cassette) can be propagated and polynucleotidesencoding products can be expressed. A host cell also includes anyprogeny of the subject host cell or its derivatives. It is understoodthat all progeny may not be identical to the parental cell since theremay be mutations that occur during replication. However, such progenyare included when the term “host cell” is used. Host cells, which areuseful in the invention, include bacterial cells, fungal cells (e.g.,yeast cells), plant cells and animal cells. For example, host cells canbe a high& eukaryotic cell, such as a mammalian cell, or a lowereukaryotic cell, such as a yeast cell, or the host cell can be aprokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology(1986)). As representative examples of appropriate hosts, there may bementioned: fungal cells, such as yeast; insect cells such as DrosophilaS2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;plant cells, and the like. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

Host cells for use in the invention are eukaryotic host cells (e.g.,mammalian cells). In one aspect of the invention the host cells aremammalian production cells adapted to grow in cell culture. Examples ofsuch cells commonly used in the industry are CHO, VERO, BHK, HeLa, CV1(including Cos; Cos-7), MDCK, 293, 3T3, C127, myeloma cell lines(especially murine), PC12 and W138 cells. Chinese hamster ovary (CHO)cells, which are widely used for the production of several complexrecombinant proteins, e.g. cytokines, clotting factors, and antibodies(Brasel et al., Blood 88:2004-2012 (1996); Kaufman et al., J. Biol Chem263: 6352-6362 (1988); McKinnon et al., J Mol Endocrinol 6:231-239(1991); Wood et al., J. Immunol 145:3011-3016 (1990)). The dihydrofolatereductase (DHFR)-deficient mutant cell lines (Urlaub et al., Proc NatlAcad Sci USA 77:4216-4220 (1980)) are the CHO host cell lines of choicebecause the efficient DHFR selectable and amplifiable gene expressionsystem allows high level recombinant protein expression in these cells(Kaufman, Meth Enzymol 185:527-566 (1990)). In addition, these cells areeasy to manipulate as adherent or suspension cultures and exhibitrelatively good genetic stability. CHO cells and recombinant proteinsexpressed in them have been extensively characterized and have beenapproved for use in clinical manufacturing by regulatory agencies. Inaddition, it is contemplated that host cells derived from any of theforegoing cell lines and having a desired phenotype may also be used.For example, a derived host cell includes CHO cells (e.g., the DG44 cellline), which have been selectively cultured for a desired phenotype(e.g., by positive and/or negative selection processes).

In one aspect of the invention, an expression system for in vitroproduction of an agent encoded by a polynucleotide of interest isprovided. As discussed herein, the polynucleotide of interest can encodea polypeptide of pharmaceutical, medicinal, nutritional, and/orindustrial value. For example, the polynucleotide of interest can encodea polypeptide-based drug. Typically such a polypeptide will be expressedas an extracellular product. For example, polypeptides that may beproduced using the expression cassette and/or vector of the inventioninclude, but are not limited to, a Flt3 ligand, a CD40 ligand,erythropoeitin, thrombopoeitin, calcitonin, Fas ligand, ligand forreceptor activator of NF-kappa B (RANKL), TNF-related apoptosis-inducingligand (TRAIL), ORK/Tek, thymic stroma-derived lymphopoietin,granulocyte colony stimulating factor, granulocyte-macrophage colonystimulating factor, mast cell growth factor, stem cell growth factor,epidermal growth factor, RANTES, growth hormone, insulin,insulinotropin, insulin-like growth factors, parathyroid hormone,interferons (e.g., interferon beta), nerve growth factors, glucagon,interleukins 1 through 18, colony stimulating factors, lymphotoxin-β,tumor necrosis factor, leukemia inhibitory factor, oncostatin-M, variousligands for cell surface molecules Elk and Hek (such as the ligands foreph-related kinases, or LERKS), and antibody light or heavy chains.

Receptors for any of the aforementioned proteins can also be expressedusing the inventive methods and compositions, including both forms oftumor necrosis factor receptor (referred to as p55 and p75),Interleukin-1 receptors (type 1 and 2), Interleukin-4 receptor,Interleukin-15 receptor, Interleukin-17 receptor, Interleukin-18receptor, granulocyte-macrophage colony stimulating factor receptor,granulocyte colony stimulating factor receptor, receptors foroncostatin-M and leukemia inhibitory factor, receptor activator ofNF-kappa B (RANK), receptors for TRAIL, BAIT receptor, lymphotoxin betareceptor, TGFβ receptor types. I and II, and receptors that includedeath domains, such as Fas or Apoptosis-Inducing Receptor (AIR).

Other proteins that can be expressed using the expression cassetteand/or vectors of the invention include cluster of differentiationantigens (referred to as CD proteins), for example, those disclosed inLeukocyte Typing VI (Proceedings of the VIth International Workshop andConference; Kishimoto, Kikutani et al., eds.; Kobe, Japan, 1996), or CDmolecules disclosed in subsequent workshops. Examples of such moleculesinclude CD27, CD30, CD39, CD40, and ligands thereto (CD27 ligand, CD30ligand and CD40 ligand). Several of these are members of the TNFreceptor family, which also includes 41BB and OX40; the ligands areoften members of the TNF family (as are 41BB ligand and OX40 ligand);accordingly, members of the TNF and TNFR families can also be expressedusing the invention.

Polypeptides that are enzymatically active can also be expressedaccording to the invention. Examples includemetalloproteinase-disintegrin family members, various kinases,glucocerebrosidase, superoxide dismutase, tissue plasminogen activator,Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, globins,an IL-2 antagonist, alpha-1 antitrypsin, TNF-alpha Converting Enzyme(TACE), and numerous other enzymes. Ligands for enzymatically activeproteins can also be expressed using the cassette and vector of theinvention.

The inventive compositions and methods are also useful for expression ofother types of recombinant proteins and polypeptides, includingimmunoglobulin molecules or portions thereof and chimeric antibodies(e.g., an antibody having a human constant region coupled to a murineantigen binding region) or fragments thereof. Numerous techniques areknown by which DNAs encoding immunoglobulin molecules can be manipulatedto yield DNAs encoding recombinant proteins such as single chainantibodies, antibodies with enhanced affinity, or other antibody-basedpolypeptides (see, for example, Larrick et al., Biotechnology 7:934-938(1989); Reichmann et al., Nature 332:323-327 (1988); Roberts et al.,Nature 328:731-734 (1987); Verhoeyen et al., Science 239:1534-1536(1988); Chaudhary et al., Nature 339:394-397 (1989)). Cloned humanizedantibodies include those specifically binding to lymphotoxin betareceptor and integrins such as VLA-1, VLA-4, and αvβ6, Such antibodiescan be agonists or antagonists.

Various fusion proteins can also be expressed using the inventivemethods and compositions. Examples of such fusion proteins includeproteins expressed as a fusion with a portion of an immunoglobulinmolecule, proteins expressed as fusion proteins with a zipper moiety,and novel polyfunctional proteins such as a fusion proteins of acytokine and a growth factor (e.g., GM-CSF and IL-3, MGF and IL-3). WO93/08207 and WO 96/40918 describe the preparation of various solubleoligomeric forms of a molecule referred to as CD40L, including animmunoglobulin fusion protein and a zipper fusion protein, respectively;the techniques discussed therein are applicable to other proteins.

Once a polynucleotide of interest is expressed, the expression product(e.g., a protein or polypeptide) may be purified using standardtechniques in the art. For example, where the polynucleotide of interestencodes a fusion polypeptide comprising a purification tag, thepolypeptide may be purified using antibodies that specifically bind tothe tag. In one aspect an oligonucleotide encoding a tag molecule isligated at the 5′ or 3′ end of a polynucleotide of interest encoding adesired polypeptide; the oligonucleotide may encode a polyHis (such ashexaHis), or other “tag” such as FLAG HA (hemaglutinin Influenza virus)or myc for which commercially available antibodies exist. This tag istypically fused to the polypeptide upon expression of the polypeptide,and can serve as means for affinity purification of the desiredpolypeptide from the host cell. Affinity purification can beaccomplished, for example, by column chromatography using antibodiesagainst the tag as an affinity matrix. Optionally, the tag cansubsequently be removed from the purified polypeptide by various meanssuch proteolytic cleavage.

The expression cassette and vectors of the invention can be used toprovide a stable transfer of a polynucleotide of interest into a hostcell. A stable transfer means that the polynucleotide of interest iscontinuously maintained in the host. The expression cassette or vectorof the invention may also provide transient expression of apolynucleotide of interest in a host cell. Transiently transfected hostcells lose the exogenous DNA during cell replication and growth.

An expression cassette of the invention may be used to deliver atherapeutic agent to a human or animal in need of treatment.Alternatively, the expression cassette of the invention may be used todeliver an agent encoding potentially immunogenic polypeptides in vivofor vaccine purposes to a subject (e.g., a human), particularly forvaccination of domesticated animals including animals of foodstock suchas fish, porcine, equine, bovine, canine, and feline species.

The expression cassette of the invention may be administered directly asa naked nucleic acid construct, typically comprising flanking sequenceshomologous to a host cell genome. Uptake of naked nucleic acidconstructs by vertebrate cells is enhanced by several known techniquesincluding biolistic transformation and lipofection.

Alternatively, the expression cassette may be administered as part of avector, including a plasmid vector or viral vector.

Typically the expression cassette or vector is combined with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions including, for example, phosphate-bufferedsaline. The composition comprising the expression cassette or vector canbe formulated for various types of administration including, forexample, intramuscular administration.

When the composition comprising the expression cassette or vector isused in an injectable form, it is typically mixed with a vehicle that ispharmaceutically acceptable for an injectable formulation for directinjection at the site to be treated. The pharmaceutically acceptablecarrier or diluent maybe, for example, a sterile isotonic solution. Thecomposition comprising the expression cassette or vector may also beformulated in an orally active form.

The actual formulation used can be readily determined by the skilledperson and will vary depending on the nature of the substance to beadministered and the route of administration.

The dose of substance used may be adjusted according to variousparameters, especially according to the substance used, the age, weightand condition of the subject to be treated, the mode of administrationused and the required clinical regimen. A physician will be able todetermine the required route of administration and dosage for anyparticular subject and condition.

Examples Construction of pV10 Vector

Additional details of the construction of pV10 are outlined in FIG. 1.Genomic DNA was isolated from human diploid fibroblasts infected withhuman cytomegalovirus strain AD169 (ATCC No. VR-538) and used as atemplate to PCR amplify the CMV immediate early gene 1 promoter/enhancerregion (CMV IE1 P/E) (see FIG. 11(B) (SEQ ID NO:1) for details of the5′UTR of the CMV IE1 gene). The promoter was amplified using primerscontaining a HindIII site at the 5′ terminus(tttAAGCTTGACATTGATTATTGACTAG; SEQ ID NO:2; restriction site underlined)and a BamHI site at the 3′ terminus (ttttGGATCCCTGTCAAGGACGGTGACTGC; SEQID NO:3; restriction site underlined). The terminal “t” nucleotidespreceding the restriction site are included in the oligonucleotidedesign to facilitate restriction enzyme digestion and are eliminated inthe cloning step.

All PCR reactions were performed in the DNA engine PTC-200 PelierThermal Cycler (MJ Research, Watertown, Mass.). The total reactionvolume was 100 μl: 1× NEB Vent polymerase buffer (10 mM KCl, 20 mM TrispH 8.8 at 25 ° C., 10 mM (NH₄)SO₄, 2 mM MgSO₄, 0.1% Triton X-100), 2.5mM dNTP's, 2 units Vent DNA polymerase (New England Biolabs, Beverly,Mass.), 1 μg of each primer, 1 μg of genomic DNA isolated from CMVinfected cells as template. The reaction conditions were as follows: 99°C. for 1 minute, 55° C. for 30 seconds, 75° C. for 1.5 minutes for 15cycles. The resulting fragment was digested with restriction enzymesBamHI and HindIII (New England Biolabs) and subcloned into the cloningvector pUC19 digested with BamHI and HindIII. The sequence analysis ofthe insert was determined and was consistent with the published sequenceof the CMV IE1 promoter/enhancer region cloned.

Construction of pV40 Vector

The construction of pV40 is outlined in FIG. 2. The 3′UTR of the hGHvgene including the polyA signal was PCR amplified from genomic DNAisolated from human fibroblasts. The (+) strand of 5′ primer(TTTTGGATCCCTGCCCGGGTGGCATCC; SEQ ID NO:20) contained a terminal BamHIrestriction site and the (−) strand or 3′ primer contained a terminalEcoRI site (TTTTGAATTCATGAGAGGACAGTGCCAAGC; SEQ ID NO:21). The PCRconditions were the same as described for the construction of pV10. Theresulting PCR fragment was digested with BamHI and EcoRI, gel purifiedand ligated into vector pV10 digested with BamHI and EcoRI. Theresulting plasmid, designated pV40, was verified by restriction enzymeanalysis. Subsequent sequencing indicated that a small number ofnucleotide differences between the 3′UTR of this hGHv gene (SEQ IDNO:19) and the published hGHv gene sequence in GenBank Accession No.K00470 (SEQ ID NO:18). At least some of the changes are due to allelicvariations.

Construction of pV70 Vector

The pV40 vector was digested with BspE1 and HpaI to remove a 317nucleotide section of the Intron A region (IVS) (see, e.g., FIGS. 2 and3). pV60 was generated by blunt end ligation into BspEI-HpaI of the dhfrcoding region of pV40. The pV70 expression vector contains the humancytomegalovirus major immediate early 1 (hCMV IE1) promoter/enhancerregion to regulate transcription. It also contains the hCMV IE1 5′UTRand intron A, where the intron contains a 317 base pair deletion. Forthe termination of transcription, the vector contains the human growthhormone variant polyadenylation (hGHv poly A) region, SEQ ID NO:19.Construction of pV40, pV60, and pV70 are detailed below and furtherdescribed in the Figures.

A. Generation of pXLC.1

A PCR product containing a light chain coding sequence for an antibodywas digested with BamHI and cloned into a unique BamHI site in theexpression vector pV70 (FIG. 3). The light chain coding region wasinserted into a unique BamHI site between the 5′ UTR sequence at the 3′end of the hCMV IE1 intron and the 5′ end of the hGHv poly A region.This plasmid was designated as pXLC.1. FIG. 4 shows a schematic of thegeneration of the pXLC.1 vector.

B. Addition of a Neo Cassette—pXLC.2

A neomycin transferase (neo) expression cassette was introduced intopXLC.1 to act as a selectable marker (FIG. 5). The neo cassette wasprepared as a BamHI/EcoRI fragment from a commercially available plasmidcalled pGT-N28 (New England Biolabs, catalog #307-28). In this plasmid,the neo gene is driven by the phosphoglycerate kinase (PGK) promoter andterminated at a PGK poly-adenylation site. The BamHI end at the 5′ endof the neo cassette was converted to a NarI end using the followingadaptor oligos:

BamHI compatible        EcoRI GATCGATGAATTCGG (SEQ ID NO: 4)CTACTTAAGCCGC (SEQ ID NO: 5) NarI compatible

With these linkers, a new EcoRI site was also added. The adaptor wasfirst ligated to the BamHI/EcoRI cut neo fragment, and the convertedNarI/EcoRI fragment was then cloned into pXLC.1 digested with NarI andEcoRI. In this way the neo expression cassette was inserted at the 3′end of the light chain sequence of the plasmid. This plasmid wasdesignated as pXLC.2 (FIG. 5).

Construction of the Heavy Chain Expression Vector—pXHC.5

A. RT-PCR of the Heavy Chain

The heavy chain was amplified from the RT-PCR reaction using the 5′ PCRprimer TTTTGGATCCATGTACTGGGTGAAGCAG (SEQ ID NO:6), where the italicizedsequence is an added linker region with a BamHI site, and the underlinedbases correspond to the second methionine in the heavy chain codingsequence. The 3′ PCR primer that was used, GCCCGGATCCTCATTTACCCGGAGACAG(SEQ ID NO:7), also contains an added linker region with a BamHI site(italics) and a sequence that corresponds heavy chain coding sequenceincluding the termination codon (underlined). The expected PCR productof 1268 base pairs was obtained.

B. Construction of pXHC

Because the 5′ PCR primer used hybridized with the second ATG codon inthe coding region rather than the initiation ATG the coding regioncontained in the PCR product, the heavy chain coding region wasincomplete. The BamHI fragment, containing the incomplete heavy chain,was cloned into the plasmid pV60 (FIG. 6). This vector is identical topV70 (described above) except that it contains the dhfr coding region atthe site of the deletion in the intron. The heavy chain coding regionwas inserted into a unique BamHI site between the 5′ untranslatedsequence at the 3′ end of the hCMV IE1 intron and the 5′ end of the hGHvvariant poly A region. This plasmid, with the incomplete heavy chain,was designated as pXHC.

C. Completion of the Heavy Chain Coding Sequence—pXHC.1

The coding region that was missing from the heavy chain sequence wasinserted into pXHC to generate the plasmid designated as pXHC.1 (FIG.7). To do this, a fragment was generated by PCR using a plasmidcontaining the coding sequence for the antibody as a template. The 5′PCR primer used was:

-   -   PstI BamHI

TTTTCTGCAGTCACCGTCCTGACACGGGATCCATGGACTGGACCTTGGAGGG (SEQ ID NO:8). Thesequence in italics corresponds to the pXHC sequence 5′ of the BamHIsite and the sequence in bold corresponds to the sequence starting twobases before the initiation codon in the heavy chain sequence. The 3′primer used was CTGAGGAGACGGTGACCAGGGTCCCTTGGCCCC (SEQ ID NO:9). Thisprimer hybridizes to the end of the first exon, the heavy chain variableregion. A PCR product of 445 base pairs was obtained as expected and cutwith PstI and StuI. The PstI/StuI fragment was cloned into pXHC cut withPstI and StuI to yield pXHC.1.

D. Removal of the Intronic dhfr—pXHC.3

pXHC.1 contained a dhfr gene in the hCMV IE1 intron. Due to potentialproblems with amplification found with this configuration, the dhfr genewas removed from the intron and an expression cassette was inserted 3′of the heavy chain cassette (FIG. 8). The first step was the removal ofthe dhfr gene from the intron. This was accomplished by cutting theheavy chain coding sequence out of pXHC.1 as a PstI/EcoRI fragment andcloning it into the pXLC.1 plasmid cut with the same enzymes. Thiscloning step simply switched the light chain for the heavy chain in theplasmid. The resulting plasmid was identical to pXHC.1 except that theintron containing the dhfr gene was replaced by an intron with adeletion as described above for pXLC.1. This heavy chain plasmid wasdesignated as pXHC.3 (FIG. 8).

E. Insertion of the dhfr Cassette—pXHC.5

The second step in the alteration of the dhfr configuration was theinsertion of the dhfr expression cassette 3′ of the heavy chainexpression cassette (FIG. 9). The dhfr expression cassette was derivedfrom the plasmid pSI-DHFR on a BglII/BamHI fragment and includes an SV40early promoter, the dhfr gene and an SV40 poly A region. This fragmentwas cloned into the EcoRI site located at the 3′ end of the hGHv poly Aregion of pXHC.3 using the following adaptor oligos:

EcoRI compatible       SalI AATTCGTCGACA (SEQ ID NO: 10) GCAGCTGTCTAG(SEQ ID NO: 11) BamHI/BglII compatibleThis adaptor was first ligated to the EcoRI cut plasmid and then theBglII/BamHI dhfr cassette was ligated to the adapted plasmid. Thisplasmid was designated as pXHC.5 (FIG. 9).

Characterization of pXLC.2 and pXHC.5

Both plasmids were analyzed using restriction enzymes to confirm thepresence and orientation of the inserted fragments. In addition, thecoding region of the plasmids was sequenced to verify that no mutationswere accumulated during the PCR or cloning steps.

The presence of a functional neo selection marker was confirmed bytransfecting pXLC2 into CHO cells and demonstrating resistance to G418.The ability to do a dual selection was demonstrated when the pXLC.2 andpXHC.5 plasmids were co-transfected into a serum free adapted DG44 CHOhost. Colonies grew out from the co-transfection in a dual selectionmedia (a-MEM 10% dFBS with 400 mg/ml 0418). Under the same conditions,either selection alone (a-MEM or 400 mg/ml G418) was able to killuntransfected cells.

Construction of the Vectors: pV80 and pV90 Vectors

pV80 was generated from the heavy chain expression vector pXHC.5 (FIG.10). The heavy chain coding sequence was deleted from pXHC.5 usingBamHI. The backbone was ligated to re-circularize it at the BamHI site.

Two alterations were made to the pV80 vector to generate pV90 (FIG. 11).The Not1 site found in the pV80 construct at position 3166 (at the endof the dhfr coding region, see attached sequence) was destroyed. Toaccomplish this, the plasmid was digested with Not1 and the overhangswere “filled-in” using Klenow polymerase. As a result, the re-ligatedplasmid had lost the NotI site at position 3166. A new NotI site wasthen created at the cloning site by digesting the vector with BamHI andintroducing a NotI linker made by annealing the following 14-mer withitself: GATCCGCGGCCGCG (SEQ ID NO:12). When annealed together, thelinker sequence is:

BamHI compatible        NotI GATCC GCGGCCGC (SEQ ID NO: 13) CGCCGGCGCCTAGG (SEQ ID NO: 14) BamHI compatible

This cloning step recreated BamHI sites on either side of the NotI site.These BamHI sites may be useful in the genetic analysis of stable celllines generated with this vector. The cloning site sequence of pV90 wasconfirmed by sequence analysis.

In the pV80 vector a polynucleotide (e.g., a coding sequence) may becloned into the BamHI site GGATCCCTGCCCGGGT (SEQ ID NO:15). The boldsequence represents the BamH1 site. In the pV90 vector a polynucleotide(e.g., a coding sequence) may be cloned into the BamHI or NotI siteGGATCC GCGGCCGC GGATCC CTGCCCGGGT (SEQ ID NO:16). Here, the boldsequence represents the BamH1 site and the underlined sequencerepresents the Not1 site. For optimal results when using the NotI site,a “C” should be added prior to the start codon in the PCR primers tobest match the Kozak sequence (e.g., GGATCC GCGGCCGC C ATG (SEQ IDNO:17)).

Restriction sites in pV80 and pV90

pV80 and pV90 are identical with the exception of Not1 restrictionsites: pV80 has a single NotI site at position 3166 (end of the dhfrcoding region) and pV90 has a single NotI site at position 1260 (cloningsite).

Common restriction sites of which 2 or fewer were found include thoselisted in Table 1.

TABLE 1 AatI(1) 2274 ApaI(1) 1733 AspEI(2) 1382, 4807 BamHI(1) 1254BsgI(1) 1494 BsiXI(1) 3404 ClaI(1) 3404 EgeI(1) 3585 HindIII(2) 2293,6059 HpaI(1) 3308 KasI(1) 3585 Kpn2I(1) 1121 KpnI(2) 1927, 3144 NarI(1)3585 NdeI(2) 254, 3637 NheI(1) 2557 NotI(1) see above PstI(2) 1231, 2331PvuI(2) 3544, 4440 SacI(2) 585, 2819 SacII(2) 673, 1258 SmaI(2) 1271,3161 SpeI(1) 20 StuI(1) 2274 XbaI(1) 3150 XhoI(1) 3127 XmaI(2) 1271,3161

The following common enzyme cut sites were not found:

TABLE 2 AatII AfaI AfeI AgeI AluI ApaLI AspHI AspI AvaI AvaII BsiWI DpnIDpnII DraI DraII DraIII EaeI EagI EarI EcoRI EcoRV FspI HaeII HaeIIIHincI HpaI NaeI NcoI NdeII NspI PacI SalI SphI XhoII XmaIII

Cloning of the Reporter Genes

The expression cassette/vector including the CMV IE1, intron A fragment,and hGHv polyA construct was compared with a commercially availableSV40-based high expression vector.

Secreted alkaline phosphatase (SEAP) was used as reporter for thecomparison of the plasmids. The SV40 based expression vector,pSEAP2-control (Cat #6052-1, Clontech), expresses SEAP under the controlof the SV40 early promoter and SV40 enhancer. The SEAP coding sequenceis followed by the SV40 late polyadenylation signal to ensure proper,efficient processing of the SEAP transcript in eukaryotic cells. Asynthetic transcription blocker (TB), composed of adjacentpolyadenylation and transcription pause sites, located upstream of theMCS reduces background transcription. The vector incorporates a numberof features that improve the sensitivity of SEAP by increasing theefficiency of SEAP expression or that enhance the utility of thevectors. These include: an improved Kozak consensus translationinitiation site; the removal of the SV40 small-t intron, which can causecryptic splicing and reduced expression in some genes and/or cell types;switching from the early to late polyadenylation signal of SV40, whichtypically causes a five-fold increase in mRNA levels; an expandedmultiple cloning site (MCS); compact plasmid size; and removal ofextraneous sequences from the 3′ untranslated region of the SEAP mRNA.Genbank accession number U89938.

In order to generate the pCMV-hGHvPA-SEAP plasmid, the SEAP codingsequence was extracted from the pSEAP2-control plasmid by PCR and clonedinto a pV110 vector. The pV110 plasmid is a derivative of pV90 (no dhfrexpression cassette, different polylinker, but otherwise the same). ThepCMV-hGHvPA-SEAP plasmid construct was verified by sequencing.

The host used for transfections was the dihydrofolate reductase (DHFR)deficient Chinese hamster ovary cell line DG44 (Urlaub et al., Cell 33,405-412 (1983)). The CMVSEAP and pSEAP2-control reporter plasmids wereco-transfected with a plasmid encoding dihydrofolate reductase (dhfr) sothat stable transfectants could be selected for (pSI-DHFR.2, FIG. 12).Each transfection contained 50 μg of a reporter plasmid and 5 μgpSI-DHFR.2. All DNA was prepared by Megaprep kit (Qiagen). Prior totransfection, DNA was ETOH precipitated, washed in 70% EtOH, dried,resuspended in HEBS (20 mM Hepes, pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mMNa₂HPO₄, 6 mM dextrose), and quantitated prior to transfection. Negativecontrols included pUC18 (ATCC No. 37253) as a reporter control and a noDNA transfection as a transfection control (Table 3).

Cells and DNA were transfected by electroporation in 0.8 ml of HEBSusing a 0.4 cm cuvette (BioRad) at 0.28 kV and 950 mF. 5E6 cells wereused for each transfection. After the electroporation pulse, the cellswere allowed to incubate in the cuvette for 5-10 min at roomtemperature. They were then transferred to a centrifuge tube containing10 ml of alpha MEM with nucleosides and 10% dFBS and pelleted at 1K RPMfor 5 min. Resuspended pellets were seeded into 6-well plates in alphaMEM without nucleosides with 10% dFBS and incubated at 36° C. with 5%CO₂ in a humidified incubator until colonies formed.

TABLE 3 Transfection Experiment Reporter plasmid DHFR plasmid No. of (50μg each) (5 μg each) Transfections pSEAP2-control pSIDHFR.2 3 pCMVSEAPpSIDHFR.2 3 pUC18 pSIDHFR.2 1 No DNA No DNA 1

Approximately 2 weeks after transfection, colonies had formed in thetransfections containing the pSIDHFR.2 plasmid only. Stabletransfectants were analyzed as either pools or isolates. The specificproductivity was assessed in assays where the medium was exchanged forfresh medium and 24 hours later the medium was sampled and the cellswere counted. The product titer was normalized for the cell number atthe end of the 24hour assay, and the productivities were expressed asSEAP activity per cell.

SEAP assay. Conditioned medium was analyzed using the Great EscAPe™ SEAPReporter System 3 (Clontech). This assay uses a fluorescent substrate todetect the SEAP activity in the conditioned medium. The kit was used ina 96 well format according to the manufacturer's instructions, with thefollowing exceptions. The assay buffer from the kit was substituted with1.5 M diethanolamine, 0.75 mM MgCl₂, 15 mM L-homoarginine, and 10%Emerald II (Cat #9761, Applied Biosystems). All standards and sampleswere diluted in fresh medium rather than the dilution buffer provided.Instead of doing one reading after 60 min, multiple reads were taken at10-20 min intervals and used to express SEAP activity as relativefluorescent units per minute (RFU/min) The emission filter used for theplate reader (Cytofluor II, PerSeptive Biosystems) was 460 nm instead ofthe recommended 449 nm.

The RFU/min values were normalized to a standard curve based on astandard provided with the kit. Because the standard provided was notquantitated, all values are relative. These relative values werenormalized to cell numbers and the incubation period to generaterelative specific productivities (SEAP activity per cell per day).

Pools. After the appearance of colonies, the cells were collected andpooled from each transfection. Pools were seeded at ˜2×10⁵ cells perwell into 6-well plates. The following day the medium was exchanged for2 ml of fresh medium. After 24 hours the cells were counted and a sampleof the medium was used to assess SEAP activity. Results from the poolassays are shown in FIG. 13.

Isolates. Isolates were obtained by “picking” colonies from thetransfection: “Picking” was accomplished by aspirating directly over acolony with a P200 Pipetman™ set at 50 ml. The aspirated colony wastransferred first to a 48 well plate and then to a 6 well plate whenthere was a sufficient number of cells. Specific productivities wereassessed in 6 well plates at near confluent to confluent cell densitiesusing the 24-hour assay described above (FIG. 14).

As summarized in FIGS. 13 and 14, an expression vector based on thecombination of a CMV IE promoter and a hGHv polyA signal domain was farsuperior to a commercially available vector that boasts of highexpression capabilities.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-13. (canceled)
 14. An expression cassette comprising: a human CMVimmediate early 1 (hCMV IE1) promoter/enhancer region; a polynucleotideof interest; and a polyA signal domain comprising SEQ ID NO:
 19. 15. Theexpression cassette of claim 14, further comprising a variable lengthintervening sequence (VLIVS) comprising a splice donor and spliceacceptor site.
 16. The expression cassette of claim 15, wherein theVLIVS comprises an intron A of a hCMV IE1 gene that has a deletionbetween the splice acceptor and splice donor of the intron A.
 17. Theexpression cassette of claim 14, further comprising an additionalpromoter/enhancer element or regulatory region.
 18. The expressioncassette of claim 17, wherein the promoter/enhancer element is an SV40promoter/enhancer element.
 19. The expression cassette of claim 17,wherein the regulatory region is an SV40 polyadenylation region.
 20. Theexpression cassette of claim 14, further comprising a selectable marker.21. The expression cassette of claim 20, wherein the selectable markeris selected from the group consisting of dihydrofolate reductase, GFP,neomycin, Hygro, Zeocin™, herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase, adeninephosphoribosyltransferase, puromycin N-acetyl transferase or adenosinedeaminase.
 22. The expression cassette of claim 21, wherein theselectable marker is dihydrofolate reductase.
 23. The expressioncassette of claim 14, wherein the polynucleotide of interest encodes atherapeutic agent.
 24. The expression cassette of claim 14, wherein thepolynucleotide of interest is in the antisense orientation with respectto the promoter.
 25. The expression cassette of claim 14, wherein thepolynucleotide of interest is a heterologous polynucleotide.
 26. Theexpression cassette of claim 14, wherein the polynucleotide of interestfurther includes one or more transcriptional control elements.
 27. Theexpression cassette of claim 26, wherein the transcriptional controlelements are selected from the group consisting of transcriptional stopsignals, polyadenylation domains and downstream enhancer elements. 28.The expression cassette of claim 14, wherein the polynucleotide ofinterest encodes a polypeptide of diagnostic or therapeutic use.
 29. Theexpression cassette of claim 14, wherein the polynucleotide of interestencodes an antigenic polypeptide for use as a vaccine.
 30. Theexpression cassette of claim 14 in the form of a naked nucleic acidconstruct.
 31. An expression vector comprising an expression cassette ofclaim
 14. 32. A host cell comprising an expression vector of claim 31.33. A method for producing a polypeptide comprising propagation of ahost cell of claim 32 and expression of the polynucleotide of interest.34. A method of delivering a therapeutic agent to an animal in need oftreatment comprising propagation of a host cell of claim 33 andexpression of the polynucleotide of interest.
 35. A method of genetherapy to an animal in need of treatment comprising propagation of ahost cell of claim 33 and expression of the polynucleotide of interest.36. A polynucleotide that specifically hybridizes to a polynucleotidesequence as set forth in SEQ ID NO: 1 from about nucleotide 1 to about1867 or a fragment thereof.